Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies

Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.


Results
Construction of a high diversity nanobody library. The cDNA was prepared using the extracted total RNA from alpaca peripheral blood lymphocytes by reverse transcription (RT). The VHH genes were amplified to produce amplicons of about 450 bp by stepwise PCR using the synthesized cDNA as a template. The primary VHH library was generated by cloning the VHH gene repertoire into the T7 select 415-1b vector, followed by in vitro packaging. Subsequently the primary library generated was amplified by the liquid lysate method. The titers of the primary and amplified library were 2.73 × 10 9 PFU/mL and 1.65 × 10 11 PFU/mL, respectively. Diversity analysis of the primary library was carried out by PCR detection of 20 random phage clones ( Supplementary  Fig. S1-A). Sequence analysis indicated the typical nanobody structure with the framework region (FR) and complementarity determining region (CDR). In addition, differences in amino acid sequences of the CDRs indicated a high diversity library ( Supplementary Fig. S2). Nanobodies displayed on the T7 phage surface were Bio-panning and characterization of DC-specific binding nanobodies. To screen nanobodies that specifically bind with DCs, three rounds of phage display bio-panning were performed, and bone marrow cells were added for depletion to reduce the possibility of non-specific binding. As shown in Fig. 3A, the ratio of phage recovery in each round increased, which was considered evidence of effective screening. However, when the next generation sequencing data was assessed, it was observed that the number of phage clones with unique sequences in each round of the recovered phage had decreased (Fig. 3B), which indicated an accumulation of specific DC-binding phages during the bio-panning process.
After three rounds of screening, 125 phage clones were randomly selected for sequencing, and 46 phage clones with unique CDR sequences were identified ( Supplementary Fig. S3). To further characterize the selected phage clones, the specificity, i.e., the ability of a phage probe to associate with its target due to the presence of a specific nanobody displayed on the surface, and the selectivity, i.e., the ability of a phage probe to discriminate its cognate target from a mixture of targets were determined. The specificity of 46 unique sequence phage clones was evaluated and is summarized in Fig. 3C. Twelve phage clones with strong affinity and specificity (phage recovery higher than 0.3%) towards chicken DCs were identified. The intact amino sequence of the VHH display on phages-16, 54, 74 and 121 were analyzed in Fig. 4A, and the amino composition of the CDRs was different between these four  www.nature.com/scientificreports/ clones. Further, the selectivity of the four phage clones was evaluated ( Fig. 4B-E). It was observed that phages-54, -74, -16 and -121 bound not only to chicken DCs but also to duck and goose DCs, however, they barely bound to the bone marrow cells, chicken embryo fibroblasts (CEF), duck embryo fibroblasts (DEF) and goose embryo fibroblasts (GEF). These results suggest that these four phages may have great specificity for binding to DCs. For further verification, nanobody binding to chicken bone marrow DCs was assessed by fluorescent microscopy (Fig. 5). No green fluorescence signal was evident in Fig. 5K which indicated that the T7-wt barely bound to the DCs. In contrast, phage-54 and phage-74 could adsorb to the DCs surface efficiently, as manifested by the strong green fluorescence spot covering the DC surface (Fig. 5C and -G). In addition, the suspected DC proteins that could interact with VHH-54 and VHH-74 were analyzed by pull-down and HPLC-MS assays ( Table 1). The fusion proteins GST-VHH-54 and GST-VHH-74 were expressed and purified ( Supplementary Fig. S4) for interaction with the DC lysate. HPLC-MS results revealed several intercellular proteins such as proteases and a couple of uncharacterized proteins, unfortunately, with almost no surface proteins being discovered ( Table 1). The voltage-dependent anion-selective channel protein which was confirmed on the plasma membrane most likely interacts with VHH-74. DC targeting induced high-level antibody. The purified T7-wt, phage-54 and phage-74 particles were detected by Western-blot and a single band of fusion protein p10B-VHH-54 and p10B-VHH-74 were obtained (Fig. 6A), which indicated a correct displaying of the nanobody on the T7 phage surface. To verify the function of DC-targeting nanobodies that act in the promotion of antigen presentation and antibody formation, groups of SPF chickens were subcutaneously injected with phage-54 and phage-74, with T7-wt as a control. The level of specific antibody against T7 phage capsid was determined using ELISA. T7 phage capsid was expressed in the E. coli system, purified ( Supplementary Fig. S5) and used to coat ELISA plates to establish a detection method. Chickens immunized with phage-54 and phage-74 developed higher levels of anti-capsid antibody than chickens administered with the T7-wt control (Fig. 6B). DC-targeting phages were able to stimulate a more rapid and efficient immune response, thus indicating the potential application of the selected nanobody in antigen delivery.

Discussion
In recent years, the use of DCs to improve the immunogenicity of an antigen has been a key strategy in the field of vaccine development 28,29 . These strategies can increase the number of antigens targeting DCs, and much progress has been made in human DC-targeting vaccines 30,31 . One of the common approaches to elicit a strong and long-lasting humoral and cellular immune response is the design of DC-targeting vaccines 32,33 . When researching and developing poultry vaccines, practical factors should be taken into consideration, including production cost and wide-scale administration, consequently, DC-targeting vaccines have become a desirable choice. Hence, we www.nature.com/scientificreports/ propose to discover chicken DC-specific binding ligands which could be used as an antigen carrier to develop DC-targeting vaccines. Antibodies can specifically target receptors that are expressed on the surface of DCs, this concept has been utilized either by conjugating antigens to mAbs as DCs surface molecules, or by genetic engineering in which the antigen is fused to different antibody fragments specific to DC receptors 34 . Nanobodies, unique antibodybinding fragments derived from camelid heavy-chain antibodies, have excellent properties including thermal and chemical stability, weak immunogenicity and high affinity 35 . Thus, a T7 phage display nanobody library was constructed by inserting the alpaca VHH antibody gene into the downstream region of the T7 phage p10B gene, so that nanobodies could be displayed on the surface of the T7 phage. Compared to other phage display systems, the T7 select phage display system is easy to use and has the capacity to display peptides of about 50 to 1200 amino acids 36 . In this study, the high-copy number vector T7 select 415-1b was used to clone the VHH gene, and the E. coli BLT5403 bacterial host was used to supply the extra p10A protein to facilitate infectious recombinant phage rescue. Twenty plaques randomly screened by PCR, showed that the VHH gene was successfully inserted into the phage genome ( Supplementary Fig. S1-A), and sequencing data revealed that the framework regions and complementarity determining regions of these VHH clones displayed the greatest difference in amino acid sequences ( Supplementary Fig. S2). A small percentage of stop codons were detected within the VHH genes such as in VHH17 ( Supplementary Fig. S2), which led to the truncated VHH protein bands being detected in the Western blot ( Supplementary Fig. S1-B). In general, considering the high diversity of the VHH library and the efficient expression of VHH on the phage surface, this T7 phage display library could be adequate for biopanning needs. T7-wt phage devoid of nanobody display was used as a negative control for each assay. Phage recovery was calculated as the ratio of recovered phage versus the input phage as follows: phage recovery (%) = (output phage/ input phage) × 100. www.nature.com/scientificreports/ Antigen is bound with antibody and targeted to a DC receptor for internalization which can accelerate antigen processing and presentation 37 . The surface of DCs contains many pattern recognition receptors (PRRs). To date four PRRs have been found, including Toll-like receptors (TLRs), Nucleotide-binding oligomerization domain-like receptors (NLRs), Retinoic acid-inducible gene I-like helicases receptors (RLRs) and C-type lectin receptors (CLRs) 38,39 . Choosing the receptor to be targeted is a great challenge in the design of an antibody-based DC targeting vaccine. Although, these receptors combine specifically with the corresponding natural ligands, it is important to explore new ligands for binding with the reported receptors or other unknown receptors. For these reasons, the constructed nanobody library was used to screen intact chicken DCs, with the expectation of discovering DC-specific binding nanobodies. Although there was obvious enrichment after three rounds of screening (Fig. 3A), a decrease in phages with unique sequences was observed (Fig. 3B). Repeat phage clones were identified by sequence analysis (Supplementary Fig. S3) and this high frequency of repetition phage clones points to the success and enrichment of the screening process. As a result, twelve DC-specific binding phage clones were obtained (Fig. 3C). Unexpectedly, four of the selected phage clones bound not only to chicken DCs but also to duck and goose DCs (Fig. 4B-E). These results indicated that the chicken, duck and goose may share the common receptors that were recognized by these selected phages.
Currently, there is little consensus as to which receptor elicits more robust MHC I or MHC II antigen presentation 40 . Effective antigen presentation results from the antigen being trafficked to subcellular compartments for processing, however, individual DC receptors will differ widely in their expression levels, internalization speeds, and downstream intracellular trafficking pathways. In any event, the efficient combination of antigen and DCs is the first step for antigen processing. The interaction between the selected phage and DCs was studied by confocal laser microscopy and revealed that phage-54 and phage-74 could efficiently combine with chicken DCs within 15 min (Fig. 5). Further, nanobody binding proteins on DCs were obtained by pull-down assay and identified by mass spectrometry (Table 1). Unfortunately, none of the previously reported receptors were discovered during the mass spectrometry analysis. However, one protein, i.e., voltage-dependent anionselective channel protein (VDAC), suspected to have a role in antigen processing was discovered. Lisanti et al. 41 have previously reported the presence of VDAC1 in a catalogue of proteins identified in caveolae. Caveolae are domains of the plasma membrane that have specific functions in the trafficking between the plasma membrane and the rest of the cell 42 . Thus, it may be presumed that the specific binding of phage-74 to the VDAC of DCs made the engulfment easier, and further, sped up processing and presentation of the antigen. This supports the more rapid and higher level of the antibody response against p10B elicited by the DC-targeting nanobodies of phage-54 and phage-74 compared to that induced by T7-wt (Fig. 6B).

Conclusion
In this study, a high diversity T7 phage display nanobody library was constructed which could be used for biopanning of chicken DC-specific binding nanobodies. The results indicated that nanobodies displayed on phage-54 and phage-74 not only efficiently bind with chicken DCs but also to duck and goose DCs. Although the exact www.nature.com/scientificreports/ nanobody recognition receptor requires further elucidation, the highly efficient affinity binding of nanobody to DCs promotes the immune response. Therefore, the nanobody displayed on phage-54 and phage-74 is a DCtargeting ligand that merits further study and application.

Methods
Ethics declarations. Animals were maintained and euthanized as per the protocol, approved by the Institutional Animal Care and Use Committee (IACUC) of the Jiangsu Academy of Agriculture Sciences (SYXK 2017-2022). All experiments were conducted in accordance with the relevant guidelines and regulations of IACUC and the Institutional Biosafety Committee at the Jiangsu Academy of Agriculture Sciences. This study is reported in accordance with ARRIVE guidelines (https:// arriv eguid elines. org).

Construction of T7 phage display nanobody library.
A T7 phage display nanobody library was constructed as previously described 43 . The nanobody was displayed as an extension of the coat protein due to an in-frame insertion of the alpaca VHH gene in the p10 gene encoding the coat protein of T7 phage, resulting in the display of less than 450 guest nanobodies on the surface of each phage particle. Briefly, anticoagulated blood samples were collected from six non-immunized young alpacas (three female and three male) and lymphocytes were isolated using the Ficol separation method 44 . Total RNA was extracted using the MiniBEST Universal RNA Extration Kit (TaKaRa) and then first stand cDNA was generated using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa). The VHH gene was amplified by the stepwise PCR method 45 and all the primers used are presented in Table S1. The VHH gene products were digested with restriction enzymes EcoRI and HindIII and ligated to the T7 select 415-1b EcoRI/HindIII vector arms (Merck, Germany) to rescue the primary phage display library. This primary T7-VHH library was amplified using the liquid lysate amplication method according to manufacturer's instruc- Table 1. List of nanobody-binding proteins identified by LC-MS. Matches-sig: proteins that were significantly higher than the spectrum match threshold; Sequences-sig: peptides that were significantly higher than the threshold; pI: calculated isoelectric point; emPAI: exponentially modified protein abundance index. www.nature.com/scientificreports/ tions. The titers of the primary and amplified library were determined by phage-plaque assay using Escherichia coli BL5403 host 46 . Nanobody displayed on T7 phage particles were detected by SDS-PAGE and Western-blot as previously reported 47 .

Isolation and validation of chicken bone marrow DCs. Marrow collected from the femurs and tibias of three
week-old specified pathogen-free (SPF) chicks was washed three times with sterile phosphate buffered saline (PBS), gently loaded in an equal volume of Histopaque-119 (Sigma, Germany), then centifuged at 250 × g for 30 min. Cells at the interface were collected and washed twice with PBS as previously described 48 . Aliquots of 2 × 10 6 cells/mL were used to seed 6-well plates containing Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1 U/mL penicillin and streptomycin, 10% fetal bovine serum (FBS, Gibco, USA), 30 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) and 25 ng/mL interleukin-4 (IL-4) (Peprotech) and incubated at 37 °C and 5% CO 2 for 6 d. Fifty percent of the medium was replaced with complete medium every two days. Cytomorphology of DCs was observed under an optic microscope during the cell differentiation process. CD11c, CD86 and MHCII expressed on the surface of DCs on the 6th day were analyzed using fluorescence-activated cell sorting (FACS) (BD FACSCalibur, FACS101) at Taizhou People's Hospital. The duck and goose bone marrow DCs were prepared similarly.
Bio-panning of DC-specific binding phages. The T7-VHH library displaying alpaca nanobody at the C-terminal of p10B protein was applied to screen the DC-specific binding nanobody 10 . Depletion selection: monocytes isolated from bone marrow were resuspended in RPMI 1640 medium supplemented with 10% FBS and the density adjusted to 1 × 10 7 cells/mL. An aliquot of the T7-VHH library containing ~ 10 10 PFU phages was diluted in blocking buffer (RPMI 1640 medium supplemented with 10% FBS + 0.5% bovine serum albumin) and transferred to 6-well plates and left for 1 h at room temperature to deplete the library of phages that adsorb to the plastic. Unbound phages were removed and tranferred to another 6-well plate containing monocytes, then incubated for one hour at room temperature to deplete monocyte-binding phages. First round: the supernatants of the monocytes plate were transferred after centrifugation (250×g for 5 min) to incubate with DCs in a 6-well plate at room temperature for 45 min. The plate was then centrifuged at 250×g for 5 min, supernatants were removed, and cells resuspended in wash buffer (RPMI 1640 medium containing 1% FBS and 0.05% Tween-20). Washes were collected and saved for titering the phage. The wash operation was repeated five times. Thereafter, the DC-bound phages were eluted by lysing the cells with CHAPS lysis buffer (2.5% w/v CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonated] in RPMI 1640 medium) and evaluated by the phageplaque assay 46 . The phages were amplified in E. coli BL21 for the next round of bio-panning. The second and third rounds were carried out according to the procedures described above, except that the incubation time was reduced to 30 min and 15 min, respectively. The VHH genes in the total eluted phage from each round of selection were amplified, and the PCR products were sent to Genepioneer Biotechnologies Co., Ltd. (Nanjing, China) for next-generation sequencing. The individual phage plaques in the eluate of the third round of selection were randomly selected for VHH gene amplification 36 , and PCR products were sequenced by Genscript Biotechnology Co., Ltd. (Nanjing, China). www.nature.com/scientificreports/ Specificity and selectivity assay. Individual phage clones identified by DNA sequencing were propagated and purified 26 for use in cell-association assays. In the specificity assay, phage particles (~ 10 6 PFU/ well) were incubated with DCs, chicken bone marrow cells and serum-treated control wells in a 96-well cell culture plate at room temperature for 15 min. Following several washes, cell-or serum-associated phages were collected by treating each well with CHAPS lysis buffer and titering in E. coli BL5403 cells. The most promising phage binders, i.e., those phages that demonstrated increased binding to DCs rather than bone marrow cells and serum components, were tested further for their ability to discriminate between different targets (selectivity assay) using a panel of different cells from chicken, duck and goose. Dendritic cells and bone marrow cells of chicken, duck and goose were prepared as previously mentioned. Chicken embryo fibroblasts (CEF), duck embryo fibroblasts (DEF) and goose embryo fibroblasts (GEF) were prepared according to the method of Zhai et al. 49 . Cells were seeded into 96-well culture plate (~ 10 4 cell/well) in a 37 °C cell culture incubator with 5% CO 2 for 1 h. Then, each phage clone (~ 10 6 PFU/well) was incubated with cells for 15 min at room temperature. Wells were washed eight times with washing buffer by centrifugation of plate at 250×g for 5 min and unbound phages in supernatant were carefully removed. To collect cell-associated phages, 25 μL of CHAPS lysis buffer was added to each well and incubated for 10 min on a shaker with gentle rocking. Aliquots of 175 μL of overnight cultured E. coli BL5403 host cells were added to the each well and incubated for 3 min at room temperature. The final mixture was spread on LB agar plates and incubated for 3-4 h in a 37 °C incubator. The phage recovery was calculated as the percent ratio of output plaque forming units to input plaque forming units. All selectivity and specificity cell-associated assays were performed in triplicate with data reported as the mean ± standard deviation.

Subcellular localization assay.
Interactions of selected phage clones with DCs were analyzed as described previously 50 . Dendritic cells at day 6 were harvested and seeded on a cell slide, and then incubated in a 37 °C incubator with 5% CO 2 until cells were ~ 70% confluent. Next, cells were incubated with 1.0 × 10 9 PFU phages of an isolated phage clone in serum-free RPMI 1640 culture medium for 15 min at 37 °C. Cells were washed five times with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After an additional three washes, cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin for 30 min at room temperature. Cells were treated with a 1:2000 dilution of Dylight Anti-T7 tag antibody (ab117595, abcam) in blocking buffer for 1 h at room temperature. Cells were washed with PBS containing 0.05% Tween-20 (PBST) and treated with a 1:1000 dilution of Phalloidin-iFluor 594 Conjugate (abs42235791, absin) for 1 h at room temperature in the dark. After washing, cover slips were applied to the slides with VECTA shield mounting medium and DAPI. Slides were visualized with a ZEISS confocal microscope (ZEISS LSM 880) at the Testing & Analysis Center at Yangzhou University. The VHH genes from phage-54 (VVH-54) and phage-74 (VVH-74) were fused with the glutathione S-transferase (GST) gene in pGEX-4T-1 vector for fusion protein expression. The purified fusion protein and immature chicken DCs (day 6) were sent to ZoonBio Biotechnology company (Nanjing, China) for in vitro pull-down and mass spectrometry assays.
Immunogenicity of T7 phage displaying DCs nanobody. The T7-wt (T7 select 415-1b), phage-54 and phage-74 were propagated in E. coli BL5403. Briefly, 50 mL of LB medium was inoculated with 500 μL of an over-night cultured E. coli BL21 and incubated with shaking (200 rpm, 2.5 h) at 37 °C to reach a density at OD 600 nm of 1.0. The E. coli BL5403 host was then infected with the phage particles at a multiplicity of infection (MOI) of 0.001 and kept shaking at 37 °C for more than 3 h until complete cell lysis was observed. DNase I and RNase A (Takara, China) were added 30 min before harvesting the offspring phages. Phage particles were recycled by the PEG-NaCl method and extracted with 0.1% Triton-X114 to remove endotoxin 26 . The purified T7-wt, phage 54 and phage 74 were detected by Western-blot, then the titer of phages was adjusted to 10 11 PFU/mL and inactivated by 0.1% (v/v) β-propiolactone. Further, the ratio of aqueous phage to the Montanide ISA 206 (Seppic France) oil adjuvant was 54:46 (V/V) to form a water-in-oil-in-water (W/O/W) blend. Thirty SPF chickens were divided into three groups, and their necks injected subcutaneously with 0.2 mL of T7-wt, phage-54 and phage-74 emulsion, respectively. Blood samples were collected after days 0, 14, 21 and 28, and T7 phage anti-capsid antibody levels were measured using ELISA. The capsid protein of T7 phage (p10B) was expressed by pET-28a-p10B vector, and the purified protein (100 ng/mL) was used for coating to establish an indirect ELISA method. A more detailed description about the ELISA protocol used is provided in the supplementary material. All chickens were maintained and euthanized as per the protocol approved by the Institutional Animal Care and Use Committee and conducted following the guidelines of the Institutional Biosafety Committee at the Jiangsu Academy of Agriculture Sciences.

Data availability
Supplementary information accompanies this paper.